EP2913399B1 - Rna-aufbereitungsverfahren - Google Patents

Rna-aufbereitungsverfahren Download PDF

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EP2913399B1
EP2913399B1 EP13848975.2A EP13848975A EP2913399B1 EP 2913399 B1 EP2913399 B1 EP 2913399B1 EP 13848975 A EP13848975 A EP 13848975A EP 2913399 B1 EP2913399 B1 EP 2913399B1
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Prior art keywords
rna
reagent
rna extraction
extraction
pcr
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French (fr)
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EP2913399A1 (de
EP2913399A4 (de
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Sotaro Sano
Shigehiko Miyamoto
Jun Tomono
Hajime Hiratsuka
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Kaneka Corp
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Kaneka Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products

Definitions

  • the present invention relates to a method for extracting undegraded RNA from a biological sample, which makes use of a reagent for RNA extraction containing an alkali metal salt and a surfactant, as further defined in the claims.
  • RNA which is one of the targets for genetic testing in these applications, is more unstable than DNA and can be easily degraded by endogenous ribonucleases contained in biological samples or when subjected to high-temperature/alkali treatment.
  • high technologies, multi-step procedures, and expensive dedicated devices and reagents for preventing degradation of RNA have been required to prepare RNA.
  • Non Patent Literature 1 the acid guanidium-phenol-chloroform (AGPC) method
  • Non Patent Literature 2 the hot phenol method
  • both the methods are not only risky because of the use of an organic solvent and a high concentration of denaturing agent for inhibiting enzymatic reactions such as nucleic acid amplification, but also require a long-term, multi-step procedures to remove these materials, which is disadvantageous in terms of cost and ease of implementation.
  • RNA Some techniques for easily preparing RNA have also been developed in which a strong chaotropic substance and a surfactant as protein denaturing agents are used to extract RNA, without using organic solvents, which extract is then directly subjected to an enzymatic reaction.
  • a method is known in which a biological sample is dissolved using guanidine thiocyanate and sarkosyl as denaturing agents, and then RNA is extracted while protecting RNA from degradation by endogenous ribonucleases (Patent Literature 1).
  • Patent Literature 1 a method is known in which a biological sample is dissolved using guanidine thiocyanate and sarkosyl as denaturing agents, and then RNA is extracted while protecting RNA from degradation by endogenous ribonucleases.
  • Patent Literature 1 a method is known in which a biological sample is dissolved using guanidine thiocyanate and sarkosyl as denaturing agents, and then RNA is extracted while protecting
  • Patent Literature 2 a nucleic acid extraction method that uses cholic acid or glycolic acid in order not to inhibit a subsequent enzymatic reaction.
  • This technique eliminates the need of steps for purifying or diluting the nucleic acid extracted from a biological sample, and the nucleic acid can be directly subjected to an enzymatic reaction such as nucleic acid amplification.
  • this technique cannot prevent RNA degradation by endogenous ribonucleases and thus does not allow undegraded RNA to be extracted.
  • nucleic acid extraction reagents and methods have further been described in US 2004/019196 , US 2006/105372 , US 2008/003575 , WO 2007/049326 , WO 2010/083844 , WO 2012/108471 , and Shirzadegan M et al., 1991, Nucleic Acids Research, 19(21): 6055 .
  • An object of the present invention is to provide a technique for preparing RNA ready for an enzymatic reaction more easily than conventional techniques.
  • the present invention relates to a reagent for RNA extraction from a biological sample, containing an alkali metal salt and a surfactant, wherein the alkali metal salt is lithium chloride, and wherein the surfactant comprises glycolic acid and deoxycholic acid.
  • the present invention also relates to a kit for RNA extraction, including the reagent.
  • the present invention further relates to a method for RNA extraction from a biological sample, including mixing the biological sample with the reagent or the kit for RNA extraction, and extracting the RNA from the biological sample.
  • the present invention furthermore relates to a method of initiating an enzymatic reaction on RNA from a biological sample, comprising the following steps: (a) performing RNA extraction from the biological sample using a reagent for RNA extraction, wherein the reagent contains an alkali metal salt and a surfactant, wherein the surfactant comprises glycolic acid and deoxycholic acid; and (b) directly mixing the RNA extract obtained in step (a), without performing further purification or dilution, as a substrate with an enzymatic reaction solution to initiate the enzymatic reaction.
  • the alkali metal salt contained in the reagent for RNA extraction to be used in this method is preferably an alkali metal halide, more preferably lithium chloride.
  • undegraded RNA that is ready for an enzymatic or chemical reaction or the like can be more easily prepared than by conventional methods.
  • Fig. 1 shows the results of RT-PCR of the RNAs extracted from cell samples.
  • an alkali metal salt and a surfactant are used to prepare undegraded RNA from a biological sample, as defined in the claims. This allows RNA ready for a subsequent enzymatic reaction to be prepared while preventing degradation of RNA by endogenous ribonucleases in the sample.
  • the biological sample used in the present invention may be anything that contains a nucleic acid and examples include, but are not limited to, animal and plant cells and tissues, biological fluids, e.g., whole blood, serum, lymph, tissue fluid, urine, semen, vaginal secretions, amniotic fluid, tears, saliva, and sweat, cell-derived vesicles such as exosomes, stool, sputum, bacteria, and viruses.
  • biological fluids e.g., whole blood, serum, lymph, tissue fluid, urine, semen, vaginal secretions, amniotic fluid, tears, saliva, and sweat
  • cell-derived vesicles such as exosomes, stool, sputum, bacteria, and viruses.
  • RNA used in the present invention may be any RNA that contains a ribonucleotide polymer and examples include, but are not limited to, messenger RNA and non-translatable RNA such as transfer RNA, ribosomal RNA, small nuclear RNA, small nucleolar RNA, and micro RNA.
  • messenger RNA and non-translatable RNA such as transfer RNA, ribosomal RNA, small nuclear RNA, small nucleolar RNA, and micro RNA.
  • RNA is prepared by a method that is based on adsorption of RNA on a solid phase carrier, or a method that is based on addition of an organic solvent, water-soluble polymer, or surfactant to insolubilize RNA.
  • these methods not only require a complicated process for purifying the treated solution, but also show variations in recovery efficiency depending on the molecular weight of RNA, in which a target RNA may not be obtained in some cases.
  • an advantage of the RNA preparation method of the present invention is that the RNA extract can be directly subjected to a subsequent analysis, and this treated solution contains all the RNA molecules originally contained in the sample, without losses.
  • the reagent for RNA extraction which is used in the method of initiating an enzymatic reaction on RNA from a biological sample (as also specified in claim 4), contains an alkali metal salt as an RNA protecting agent.
  • Examples of the alkali metal salt used in this method of the present invention include lithium salts, sodium salts, potassium salts, rubidium salts, and cesium salts.
  • Preferred examples include, but are not limited to, lithium chloride, lithium bromide, lithium iodide, lithium acetate, lithium hydroxide, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, sodium acetate, sodium pyrophosphate, sodium thiocyanate, sodium sulfate, sodium sulfite, sodium disulfite, sodium dihydrogen phosphate, sodium hydrogen carbonate, sodium tartrate, sodium nitrate, potassium fluoride, potassium chloride, potassium bromide, potassium iodide, potassium acetate, potassium hydrogen phosphate, potassium sulfate, rubidium fluoride, rubidium chloride, rubidium bromide, rubidium iodide, rubidium acetate, cesium chloride, cesium bromide, cesium io
  • the reagent for RNA extraction provided in accordance with the present invention contains an alkali metal salt which is lithium chloride.
  • the alkali metal salt has the advantage that it is less likely to affect enzymatic activity than other metal salts that generate polyvalent cations which can be cofactors of nucleic acid-related enzymes because the alkali metal salt does not form such a cofactor.
  • the concentration is preferably at least 0.2 M but not higher than the saturated concentration, more preferably at least 0.3 M but not higher than 5.0 M, and still more preferably at least 0.3 M but not higher than 2.5 M. If the alkali metal salt concentration is too low, RNA cannot be protected from degradation by endogenous ribonucleases; if the concentration is too high, RNA can be protected but the alkali metal salt tends to inhibit a subsequent enzymatic reaction.
  • the reagent for RNA extraction contains a surfactant as an analyte solubilizer, wherein the surfactant comprises glycolic acid and deoxycholic acid.
  • surfactants described herein include ionic surfactants, non-ionic surfactants, and amphoteric surfactants, particularly Tween series surfactants such as Tween 20, Tween 40, Tween 60, and Tween 80, Triton series surfactants such as Triton X-100, Triton X-114, and Triton XL-80N, Nonidet series surfactants, non-ionic surfactants such as NP-40, and anionic surfactants such as cholic acid or its derivatives, deoxycholic acid and glycolic acid Surfactants contained in the reagent for RNA extraction are deoxycholic acid, and glycolic acid, which are used in combination.
  • the concentration is preferably not lower than 1 mM. If the surfactant concentration is too low, the biological sample tends not to be dissolvable in the reagent.
  • the reagent for RNA extraction may contain a buffer.
  • buffers that can be used in the present invention include phosphate buffers and Good's buffers. Preferred among these are Good's buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Tris, Bicine, TAPS, CHES, CAPSO, and CAPS, with TAPS being particularly preferred. These buffers may be used alone or in combination.
  • the reagent for RNA extraction may contain a protein component such as albumin, or polyamine, cyclodextrin, trehalose, a water-soluble polymer such as polyvinylpyrrolidone (PVP) or polyethylene glycol, which have an effect in reducing inhibition of enzymatic reactions by unnecessary components originating from a biological sample.
  • a protein component such as albumin, or polyamine, cyclodextrin, trehalose
  • a water-soluble polymer such as polyvinylpyrrolidone (PVP) or polyethylene glycol
  • the reagent for RNA extraction may contain an antifreeze, such as glycerol, betaine, or sucrose, to prevent freezing at sub-zero temperatures.
  • an antifreeze such as glycerol, betaine, or sucrose
  • the reagent for RNA extraction may contain an agent a chelating agent, a nuclease inhibitor, or a reducing agent (e.g. DTT (dithiothreitol)) for further ensuring inactivation of nucleases, and an organic solvent (e.g. DMSO or formamide) for a subsequent enzymatic reaction.
  • a chelating agent e.g. DTT (dithiothreitol)
  • a reducing agent e.g. DTT (dithiothreitol)
  • an organic solvent e.g. DMSO or formamide
  • the biological sample and the reagent for RNA extraction are preferably mixed at a ratio of 9:1 to 1:999, more preferably 4:1 to 1:499, and still more preferably 1:1 to 1:99.
  • the biological sample may be physically disrupted in the RNA extraction.
  • the RNA extraction may be combined with freeze-thaw disruption, physical disruption with a homogenizer, or the like.
  • RNA can be extracted without the need of disruption prior to mixing the biological sample and the reagent for RNA extraction.
  • heat treatment may be carried out to extract RNA from the biological sample.
  • the heat treatment temperature is preferably at least 0°C but not higher than 100°C, more preferably at least 30°C but not higher than 90°C, and still more preferably at least 50°C but not higher than 80°C.
  • the period of the heat treatment is preferably not longer than 30 minutes, more preferably not longer than 15 minutes. If the treatment period is too short, extraction efficiency tends to be reduced; if the treatment period is too long, RNA tends to degrade.
  • the RNA extract can be directly mixed as a substrate with an enzymatic reaction solution to initiate an enzymatic reaction such as nucleic acid amplification, without further performing purification, dilution or other steps.
  • enzymes that can be used for the reaction include nucleases such as deoxyribonucleases, ribonucleases, exonucleases, and endonucleases; proteases such as proteinases and peptidases; polymerases such as DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent RNA polymerases, heat resistant polymerases, strand-displacement polymerases, and terminal transferases; and ligases, recombinases, lysozymes, and cellulases.
  • the RNA extract and the enzymatic reaction solution are preferably mixed at a ratio of 1:999 to 999:1, more preferably 1:99 to 99:
  • the RNA extract may be mixed with a fluorescent nucleic acid labelling reagent or a fluorescent labelling probe (e.g. ethidium bromide, SYBR(R) Green, PicoGreen (R)) to detect the RNA contained therein.
  • a fluorescent nucleic acid labelling reagent e.g. ethidium bromide, SYBR(R) Green, PicoGreen (R)
  • the RNA extract may be subjected to a nucleic acid amplification reaction in the presence of a fluorescent labelling reagent to monitor the amplification reaction in real time.
  • the RNA extract may be subjected to a sequencing reaction.
  • nucleic acid amplification reaction refers to a technique for amplifying a nucleic acid sequence, as typified by PCR.
  • PCR ligase chain reaction
  • SDA strand displacement amplification
  • RCA rolling circle amplification
  • CPT cycling probe technology
  • ICAN isothermal and chimeric primer-initiated amplification of nucleic acids
  • LAMP loop-mediated isothermal amplification of DNA
  • NASBA nucleic acid sequence-based amplification method
  • TMA transcription mediated amplification method
  • the target nucleic acid in the RNA extract may be mixed with a molecule having a specific binding ability, e.g. , a molecule containing a nucleic acid sequence complementary to a part of the target nucleic acid, an antibody, or a single-stranded nucleic acid-binding protein, to specifically bind to each other; in other words, the RNA extract may be used in, for example, Southern blotting, Northern blotting, real-time PCR, or specific labelling, detection, purification or isolation using a labeled nucleic acid probe or the like.
  • a molecule having a specific binding ability e.g., a molecule containing a nucleic acid sequence complementary to a part of the target nucleic acid, an antibody, or a single-stranded nucleic acid-binding protein, to specifically bind to each other; in other words, the RNA extract may be used in, for example, Southern blotting, Northern blotting, real-time PCR, or specific labelling,
  • the RNA extract may be subjected to various chromatography techniques such as ion exchange column chromatography or gel permeation column chromatography, centrifugation, filtration, dialysis, or adsorption on a solid phase carrier to remove unnecessary components and thereby purify the RNA in the RNA extract. These techniques may also be appropriately combined for use as a RNA purification kit.
  • the kit of the present invention contains the reagent for RNA extraction.
  • the kit may contain, for example, a sample washing solution, a deoxyribonuclease, a protease, a reverse transcriptase, a DNA polymerase and its substrate, and an oligonucleotide.
  • the reagent and kit of the present invention can also be incorporated into a nucleic acid preparation device, a nucleic acid amplification device, a nucleic acid autoanalyzer, or the like.
  • Reagents for RNA extraction having the following compositions were prepared: lithium chloride, 75 mM TAPS (pH8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, and 0.05% Triton X-100.
  • the reagents for RNA extraction were prepared to have different lithium chloride concentrations of 0 M, 0.1 M, 0.2 M, 0.5 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, and 7.0 M.
  • Blood was collected from a mouse.
  • the anticoagulant used was heparin.
  • the reagents for RNA extraction (18 ⁇ l each) were individually added to 2 ⁇ l portions of the mouse blood, and incubated at 75°C for 5 minutes. After incubation, the solutions were cooled to room temperature, mixed with 2 ⁇ l (corresponding to 10 Units) of DNase I, and incubated at 42°C for 5 minutes and then at 75°C for 10 minutes.
  • RNA samples prepared above were cooled to room temperature, and RT-PCR was performed using the samples as templates to obtain an amplified nucleic acid fragment derived from the analyte.
  • the target of RT-PCR was H3F3A mRNA, and the primer set used was Forward Primer F1 (5'-GGCCTCACTTGCCTCCTGCAA-3'; SEQ ID NO:1) and Reverse Primer R1 (5'-GCAAGAGTGCGCCCTCTACTG-3'; SEQ ID NO: 2).
  • the RT-PCR was carried out using PrimeScript One Step RT-PCR Kit Ver.2 (Takara Bio, Inc.), and the RNA samples prepared above were individually added in an amount of 2% by volume to a reaction solution and subjected to the RT-PCR.
  • the reaction consisted of reverse transcription at 50°C for 30 minutes, and inactivation of the reverse transcriptase at 94°C for 1 minute, followed by 30 PCR cycles of: 94°C for 15 seconds; 62°C for 15 seconds; and then 72°C for 15 seconds.
  • 30 PCR cycles of: 94°C for 15 seconds; 60°C for 15 seconds; and then 72°C for 15 seconds were also performed without performing reverse transcription, to confirm that the amplified fragment was not derived from DNA.
  • the RT-PCR products were subjected to common agarose gel electrophoresis to visualize the amplified fragment.
  • Table 1 shows the results of visualization of the amplified fragment by agarose gel electrophoresis. The results demonstrate that in the case of the reagents for RNA extraction with a lithium chloride concentration of 0.5 to 5.0 M, only the RT-PCR produced an amplified fragment. This indicates that the RNA was properly extracted.
  • Concentration of LiCl (M) 0 0.1 0.2 0.5 1.0 2.0 3.0 4.0 5.0 6.0 7.0 RT-PCR - - - + + + + + + + + - - +: amplified -: not amplified
  • Reagents for RNA extraction having the following compositions with different alkali metal salts were prepared: 0.7 M alkali metal salt, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, and 0.05% Triton X-100.
  • the alkali metal salts used were lithium chloride, lithium bromide, lithium iodide, sodium chloride, sodium iodide, potassium chloride, potassium iodide, rubidium chloride, cesium chloride, lithium acetate, sodium dihydrogen phosphate, sodium tartrate, sodium nitrate, potassium acetate, potassium hydrogen phosphate, and potassium sulfate.
  • RNA extraction containing lithium chloride as an alkali metal salt and free from the surfactant deoxycholic acid was prepared.
  • RT-PCR was performed using the RNA samples extracted above as templates to obtain an amplified nucleic acid fragment derived from the analyte.
  • Table 2 shows the presence or absence of amplification visualized by agarose gel electrophoresis.
  • the results demonstrate that in the case of the reagents for RNA extraction containing an alkali metal salt, only the RT-PCR produced an amplified fragment. This indicates that the RNA was properly extracted.
  • the amplification efficiency of RT-PCR was high. The results further demonstrate that these reagents for RNA extraction did not inhibit the DNase I treatment, RT reaction, and PCR.
  • Reagents for RNA extraction having the following compositions with different strong chaotropic substances or polyvalent metal salts or an ammonium salt were prepared: 0.7 M strong chaotropic substance, polyvalent metal salt or ammonium salt, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, and 0.05% Triton X-100.
  • the strong chaotropic substances used were guanidine thiocyanate, guanidine hydrochloride, and urea.
  • the polyvalent metal salts used were magnesium chloride, calcium chloride, nickel chloride, and manganese chloride.
  • the ammonium salt used was diammonium hydrogen phosphate. Another reagent for RNA extraction containing the surfactants alone, without the RNA protecting agent salts, was prepared.
  • RT-PCR was performed using the RNA samples extracted above as templates.
  • Table 2 shows the results of visualization of the amplified fragment by agarose gel electrophoresis.
  • the results demonstrate that in the case of using the polyvalent metal salts, neither the RT-PCR nor PCR produced an amplified fragment. This indicates that the polyvalent metal ions did not function as RNA protecting agents, or inhibited the enzymatic reactions. Accordingly, the polyvalent cations are not preferred as additives for the reagents for RNA extraction in the present system.
  • the results also demonstrate that in the case of using the strong chaotropic substances or ammonium salt, both the RT-PCR and PCR produced an amplified fragment. This indicates that the strong chaotropic substances and ammonium salt inhibited the DNase I treatment.
  • the strong chaotropic substances and ammonium salt are not preferred as additives for the reagents for RNA extraction in the present system. It is demonstrated that in the case of containing no RNA protecting agent salt, RNA was degraded and neither the RT-PCR nor PCR produced an amplified fragment.
  • a reagent for RNA extraction having the following composition and free from surfactants was prepared: 0.7 M lithium chloride, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , and 50 mM EDTA.
  • RT-PCR was performed using the RNA sample extracted above as a template.
  • Table 2 shows the results of visualization of the amplified fragment by agarose gel electrophoresis. The results demonstrate that in the case of the reagent for RNA extraction free from surfactants, both the RT-PCR and PCR produced an amplified fragment. This is because the analyte was not completely dissolved because of the absence of surfactants, and the DNA could not be completely removed by DNase I.
  • a reagent for RNA extraction having the following composition with lithium chloride was prepared: 0.7 M lithium chloride, 75 mM TAPS (pH 8.0), 2.25 mM CaCl 2 , 15 mM MgCl 2 , 175 mM glycolic acid, 5 mM deoxycholic acid, 50 mM EDTA, and 0.05% Triton X-100.
  • the cultured cells in an amount of 10 3 to 10 5 cells were centrifuged and collected as pellets.
  • To the cell pellets was added 18 ⁇ l of the reagent for RNA extraction, and the resulting solution was incubated at 75°C for 5 minutes. After incubation, the solution was cooled to room temperature and mixed with 2 ⁇ l (corresponding to 10 Units) of DNase I, and the mixture was incubated at 42°C for 5 minutes and then at 75°C for 10 minutes.
  • RNA samples prepared as above were cooled to room temperature, and RT-PCR was performed using the samples as templates to obtain amplified nucleic acid fragments derived from the respective cells.
  • the target of RT-PCR was ACTB mRNA, and the primer set used was Forward Primer F2 (5'-AGATGGCCACGGCTGCT-3'; SEQ ID NO:3) and Reverse Primer R2 (5'-AACCGCTCATTGCCAATGG-3'; SEQ ID NO:4).
  • the RT-PCR was carried out using PrimeScript One Step RT-PCR Kit Ver.2 (Takara Bio, Inc.), and the RNA samples prepared above were individually added in an amount of 2% by volume to a reaction solution and subjected to the RT-PCR.
  • the reaction consisted of reverse transcription at 50°C for 30 minutes, and inactivation of the reverse transcriptase at 94°C for 1 minute, followed by 30 PCR cycles of: 94°C for 15 seconds; 60°C for 15 seconds; and then 72°C for 15 seconds.
  • 30 PCR cycles of: 94°C for 15 seconds; 60°C for 15 seconds; and then 72°C for 15 seconds were performed without performing reverse transcription.
  • the RT-PCR products were subjected to common agarose gel electrophoresis to visualize the amplified fragments.
  • Fig. 1 shows the amplified fragments visualized by agarose gel electrophoresis. The results demonstrate that in both the case of the HEK293T cell sample and the Jurkat cell sample, a specific amplified fragment was produced. This indicates that the respective RNAs were properly extracted.

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Claims (6)

  1. Ein Reagenz zur RNA-Extraktion aus einer biologischen Probe, umfassend:
    Lithiumchlorid; und
    ein grenzflächenaktives Mittel, das Glycolsäure und Desoxycholsäure umfasst.
  2. Ein Kit zur RNA-Extraktion, umfassend das Reagenz gemäß Anspruch 1.
  3. Ein Verfahren zur RNA-Extraktion aus einer biologischen Probe, umfassend:
    Mischen der biologischen Probe mit dem Reagenz gemäß Anspruch 1 oder einem Kit zur RNA-Extraktion, welches das Reagenz umfasst; und
    Extrahieren der RNA aus der biologischen Probe.
  4. Ein Verfahren zum Einleiten einer enzymatischen Reaktion an RNA aus einer biologischen Probe, umfassend:
    (a) Durchführen einer RNA-Extraktion aus der biologischen Probe unter Verwendung eines Reagenzes zur RNA-Extraktion, wobei das Reagenz ein Alkalimetallsalz und ein grenzflächenaktives Mittel enthält, wobei das grenzflächenaktive Mittel Glycolsäure und Desoxycholsäure umfasst; und
    (b) direktes Mischen des in Schritt (a) erhaltenen RNA-Extrakts, ohne Durchführung weiterer Reinigung oder Verdünnung, als ein Substrat mit einer enzymatischen Reaktionslösung, um die enzymatische Reaktion einzuleiten.
  5. Das Verfahren gemäß Anspruch 4, wobei das im Reagenz zur RNA-Extraktion enthaltene Alkalimetallsalz ein Alkalimetallhalogenid ist.
  6. Das Verfahren gemäß Anspruch 4, wobei das Reagenz zur RNA-Extraktion das Reagenz gemäß Anspruch 1 ist.
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JP2016182112A (ja) * 2015-03-25 2016-10-20 東ソー株式会社 ウイルスからの核酸抽出試薬
CN112176029B (zh) * 2020-06-12 2021-05-14 中山大学达安基因股份有限公司 一种拭子核酸样本释放剂及其应用

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7148343B2 (en) * 2001-10-12 2006-12-12 Gentra Systems, Inc. Compositions and methods for using a solid support to purify RNA
JP4228688B2 (ja) * 2002-12-25 2009-02-25 和光純薬工業株式会社 Ｄｎａ遊離方法
JP2004000922A (ja) * 2003-03-26 2004-01-08 Toyobo Co Ltd 核酸またはタンパク質抽出用シリカ粒子組成物
JP4836795B2 (ja) 2003-05-19 2011-12-14 ブランデイズ ユニバーシティー 核酸プロセシング方法、キット、及び装置
AU2005305012C1 (en) * 2004-11-05 2012-07-19 Qiagen North American Holdings, Inc. Compositions and methods for purifying nucleic acids from stabilization reagents
WO2007049326A1 (ja) * 2005-10-24 2007-05-03 Wako Pure Chemical Industries, Ltd. 核酸抽出方法および核酸抽出キット
US20100035331A1 (en) * 2006-02-15 2010-02-11 Tosoh Corporation Method for extracting of nucleic acid from biological material
WO2007116450A1 (ja) 2006-03-30 2007-10-18 Biocosm Inc. 核酸抽出方法及び核酸抽出キット
US20080003575A1 (en) * 2006-06-28 2008-01-03 Sigma-Aldrich Co. Methods and composition for RNA extraction
WO2010083844A1 (en) * 2009-01-26 2010-07-29 Quantibact A/S Methods and uses for rna extract and storage
JP6128371B2 (ja) * 2011-02-10 2017-05-17 Ｂｉｏｃｏｓｍ株式会社 核酸抽出液の製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

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US20150376600A1 (en) 2015-12-31
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US9695413B2 (en) 2017-07-04
CN105143449B (zh) 2018-11-13
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JP6301260B2 (ja) 2018-03-28
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